Fusion Protein Delivery System and Uses Thereof

ABSTRACT

The present invention provides a composition of matter, comprising: DNA encoding a viral Vpx protein fused to DNA encoding a protein. In another embodiment of the present invention, there is provided a composition of matter comprising: DNA encoding a viral Vpr protein fused to DNA encoding a protein. The present invention further provides DNA, vectors and methods for expressing a lentiviral pol gene in trans, independent of the lentiviral gag-pol. A gene transduction element is optionally delivered to a lentiviral vector according to the present invention. Also provided are various methods of delivering a virus inhibitory molecule to a target in an animal. Further provided is a pharmaceutical composition.

CROSS-REFERENCE TO RELATED APPLICATIONS

The patent application is a continuation of U.S. patent application Ser.No. 11/894,223, filed on Aug. 20, 2007, which is currently pending. U.S.patent application Ser. No. 11/894,223 is a continuation of U.S. patentapplication Ser. No. 10/245,475, filed Sep. 17, 2002 which is acontinuation of U.S. patent application Ser. No. 09/460,548, filed Dec.14, 1999 which is a continuation-in-part of U.S. patent application Ser.No. 09/089,900, filed Jun. 3, 1998, all of which are incorporated hereinby reference in their entirety.

FIELD OF THE INVENTION

The present invention relates generally to the fields of molecularvirology and protein chemistry. More specifically, the present inventionrelates to the use of Human and Simian Immunodeficiency Virus (HIV/SIV)Gag proteins, or amino acid residues that mediate their packaging, asvehicles for delivery of proteins/peptides to virions or virus-likeparticles and uses thereof.

DESCRIPTION OF THE RELATED ART

Unlike simple retroviruses, human and simian immunodeficiency viruses(HIV/SIV) encode proteins in addition to Gag, Pol, and Env that arepackaged into virus particles. These include the Vpr protein, present inall primate lentiviruses, and the Vpx protein, which is unique to theHIV-2/SIV_(SM)/SIV_(MAC) group of viruses. Since Vpr and Vpx are presentin infectious virions, they have long been thought to play importantroles early in the virus life cycle. Indeed, recent studies of HIV-1have shown that Vpr has nucleophilic properties and that it facilitates,together with the matrix protein, nuclear transport of the viralpreintegration complex in nondividing cells, such as the macrophage.Similarly, Vpx-deficient HIV-2 has been shown to exhibit delayedreplication kinetics and to require 2-3 orders of magnitude more virusto produce and maintain a productive infection in peripheral bloodmononuclear cells. Thus, both accessory proteins appear to be importantfor efficient replication and spread of HIV/SIV in primary target cells.

Incorporation of foreign proteins into retrovirus particles haspreviously been reported by fusion with Gag. The yeast retrotransposonTyl was tested as a retrovirus assembly model to interfere with viralreplication (Natsoulis et al. (1991) Nature 352:632-5). More recently,the expression of a murine retrovirus capsid-staphylococcal nucleasefusion protein was found to inhibit murine leukemia virus replication intissue culture cells. The expression of Gag-staphylococcal nucleasereduces viral titer and diminishes viral infectivity to promote ananti-HIV strategy (Schumann et al. (1996) J. Virol. 70:432937).

Lentiviral vectors, specifically those based on HIV-1, HIV-2 and SIV,have utility in gene therapy, due to their attractive property of stableintegration into nondividing cell types (Naldini et al. (1996) Science272:263-267; Stewart et al. (1997) J. Virol. 71:5579-5592; Zhang et al.(1993) Science 259:234-238). The utility of lentiviral-based vector usefor human therapy requires the development of a safe lentiviral-basedvector. HIV virion associated accessory proteins (Vpr and Vpx) have beenshown to be useful as vehicles to deliver protein of both viral andnon-viral origin into HIV particles (Liu et al. (1995) J. Virol.69:7630-7638; Liu et al. (1997) J. Virol. 71:7704-7710; Wu et al. (1994)J. Virol. 68:6161-6169; Wu et al. (1997) EMBO Journal 16:5113-5122; Wuet al. (1996) J. Virol. 70:3378-3384). We recently demonstrated thattrans-RT and IN mimic cis-RT and IN (derived from Gag-Pol). The trans-RTand IN proteins effectively rescue the infectivity and replication ofvirions derived from RT-IN minus provirus through the complete lifecycle (Liu et al. (1997) J. Virol. 71:7704-7710 and Wu et al. (1994) J.Virol. 68:6161-6169). Moreover, these findings demonstrate thattruncated Gag-Pol precursor polyprotein (Gag-Pro) support the formationof infectious particles when the functions of RT and IN are provided intrans. This finding demonstrated for the first time for a lentivirusthat the full length Gag-Pol precursor is not required for the formationof infectious particles. Our data also show that trans Vpr-RT-IN, orVpr-RT together with Vpr-IN are fully functional and support virusinfectivity, integration of the proviral DNA, and replication (throughone cycle) of RT defective, IN defective and RT-IN defective viruses(Liu et al. (1997) J. Virol. 71:7704-7710 and Wu et al. (1994) J. Virol.68:6161-6169). It should also be noted that our data demonstrate thatenzymatically active RT does not require Vpr for incorporation intovirions (FIGS. 19A and B). RT can be incorporated into HIV-1 virionswhen expressed in trans even without its expression as a fusion partnerof Vpr. These data demonstrate that the functions of these criticalenzymes can be provided in trans, independent of their normal mechanismfor expression and virion incorporation as components of the Gag-Polprecursor protein.

The prior art is deficient in the lack of effective means of deliveringor targeting foreign, e.g., toxic proteins to virions. The presentinvention fulfills this longstanding need and desire in the art.

SUMMARY OF THE INVENTION

The present invention shows that Vpr and Vpx can be used as vehicles totarget foreign proteins to HIV/SIV virons. Vpr1 and Vpx2 gene fusionswere constructed with bacterial staphylococcal nuclease (SN) andchloramphenicol acetyl transferase (CAT) genes. Unlike Gag or Polproteins, Vpr and Vpx are dispensable for viral replication inimmortalized T-cell lines. Thus, structural alteration of theseaccessory proteins may be more readily tolerated than similar changes inGag or Gag/Pol. Fusion proteins containing a Vpx or Vpr moiety should bepackaged into HIV particles by expression in trans, since theirincorporation should be mediated by the same interactions with Gag thatfacilitates wild-type Vpr and Vpx protein packaging.

Vpr and Vpx fusion proteins were constructed and their abilities topackage into HIV particles were demonstrated. Fusion partners selectedfor demonstration were: staphylococcal nuclease because of its potentialto degrade viral nucleic acid upon packaging and chloramphenicol acetyltransferase because of its utility as a functional marker. To controlfor cytotoxicity, an enzymatically inactive nuclease mutant (SN*),derived from SN by site-directed mutagenesis was also used. This SN*mutant differs from wild-type SN by two amino acid substitutions; Gluwas changed to Ser (position 43) and Arg was changed to Gly (position87). SN* folds normally, but has a specific activity that is 10⁶-foldlower than wild-type SN. Using transient expression systems and in transcomplementation approaches, fusion protein stability, function andpackaging requirements was shown. The present invention shows that Vpr1and Vpx2 fusion proteins were expressed in mammalian cells and wereincorporated into HIV particles even in the presence of wild-type Vprand/or Vpx proteins. More importantly, however, the present inventionshows that virion incorporated Vpr and Vpx fusion remains enzymaticallyactive. Thus, targeting heterologous Vpr and Vpx fusion proteins,including deleterious enzymes, to virions represents a new avenue towardanit-HIV drug discovery.

In one embodiment of the present invention, there is provided acomposition of matter, comprising: DNA encoding a viral Vpx proteinfused to DNA encoding a virus inhibitory protein.

In another embodiment of the present invention, there is provided acomposition of matter, comprising: DNA encoding a viral Vpr proteinfused to DNA encoding a virus inhibitory protein.

The present invention shows that Gag and/or Gag variants can be used asvehicles to target proteins of viral and non-viral origin into HIV/SIVvirions. Gag gene fusions were constructed with bacterial staphylococcalnuclease (SN), chloramphenicol acetyl transferase (CAT) genes, greenfluorescence protein (GFP), reverse transcriptase (RT), integrase (IN)and combinations thereof. Fusion proteins containing a Gag moiety shouldbe packaged into HIV particles by expression in trans, to the nativeviral genome.

Gag fusion proteins were constructed and their abilities to package intoHIV particles were demonstrated. The present invention shows that Gagfusion proteins were expressed in mammalian cells and were incorporatedinto HIV particles even in the presence of wild-type Gag proteins. Thepresent invention further shows that virion incorporated Gag fusionsremain infective in contrast to the prior art (Schuman et al. (1996) J.Virol. 70:4379-37). Thus, targeting heterologous Gag fusion proteins,including deleterious enzymes, to virions represents a new avenue towardanti-HIV drug discovery and gene therapy.

The invention shows that Gag proteins and variants thereof are operativeas vehicles to deliver fully functional RT and IN in trans intolentiviral and retroviral particles, independently of their normalexpression as components of the Gag-Pol precursor protein. Thereforethis invention generates a novel packaging component (Gag-Pro), and anovel trans-enzymatic element that provides enzyme function forretroviral-based vectors. According to the present invention, thegeneration of potentially infectious/replicating retroviral forms(LTR-gag-pol-LTR) is decreased, since according to the present inventionthis requires recombination of at least three separate RNAs derived fromthe different plasmids: vector plasmid, packaging plasmid, atrans-enzyme expression plasmid and envelope plasmid, and as such isunlikely to occur. Virion Gag proteins are utilized in the presentinvention as vehicles to deliver the RT and IN proteins into lentiviralvectors, independently of Gag-Pol. As such, a “trans-lentiviral” or“transretroviral” vector is utilized for gene delivery, and genetherapy.

In one embodiment of the present invention, there is provided acomposition of matter, comprising: DNA encoding a viral Gag proteinfused to DNA encoding a virus inhibitory protein.

In another embodiment of the present invention, there is provided acomposition of matter, comprising: DNA encoding a viral Gag proteintruncate fused to DNA encoding a virus inhibitory protein.

In yet another embodiment of the present invention, there is provided amethod of delivering a virus inhibitory molecule to a target in ananimal, comprising the step of administering to said animal an effectiveamount of the composition of the present invention.

In still yet another embodiment of the present invention, there isprovided a pharmaceutical composition, comprising a composition of thepresent invention and a pharmaceutically acceptable carrier.

Other and further aspects, features, and advantages of the presentinvention will be apparent from the following description of thepresently preferred embodiments of the invention given for the purposeof disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

So that the matter in which the above-recited features, advantages andobjects of the invention, as well as others which will become clear, areattained and can be understood in detail, more particular descriptionsof the invention briefly summarized above may be had by reference tocertain embodiments thereof which are illustrated in the appendeddrawings. These drawings forth a part of the specification. It is to benoted, however, that the appended drawings illustrate preferredembodiments of the invention and therefore are not to be consideredlimiting in their scope.

FIG. 1 shows the construction of vpr1, vpr1SN/SN*, vpx2 and vpx2SN/SN*expression plasmids. FIG. 1A shows the illustration of the pTM-vpr1expression plasmid. The HIV-1_(YU2) vpr coding region was amplified byPCR and ligated into pTM1 at the NcoI and BamHI restriction sites. FIG.1B shows the illustration of the pTM-vpx2 expression plasmid. TheHIV-2_(ST) vpx coding region was amplified by PCR and ligated into pTM1at the NcoI and Bg1 II/SmaI sites. FIG. 1C shows the illustration of thefusion junctions of the pTM-vpr1 SN/SN* expression plasmids (SEQ ID NO:11 and 12). SmaI/XhoI DNA fragments containing SN and SN* were ligatedinto HpaI/XhoI cut pTM-vpr1. Blunt-end ligation at HpaI and SmaI siteschanges the vpr translational stop codon (TAA) to Trp and Substitutedthe C terminal Ser with a Cys residue. FIG. 1D shows the illustration ofthe fusion junctions of the pTM-vpx2SN/SN* expression plasmids (SEQ IDNO:13 and 14). BamHI/XhoI DNA fragments containing SN and SN* wereligated into BamHI/XhoI cut pTM-vpx2. In the construction of theseplasmids, the Vpx C terminal Arg codon was changed to a Val codon and aSer residue was introduced in place of the Vpx translational stop codon(TAA). Fusion of vpx and SN/SN* at the BamHI sites left a short aminoacid sequence of the pTM1 polylinker (double underlined) between the twocoding regions.

FIG. 2 shows the expression of Vpr1- and Vpx2-SN and SN* fusion proteinsin mammalian cells. FIG. 2A shows the pTM1, pTM-vpr1, pTM-vpr1SN andpTM-vpr1 SN* were transfected into HeLa cells one hour after infectionwith rVT7 (MOI=10). Twenty-four hours later cell lysates were preparedand examined by immunoblot analysis. Replica blots were probed withanti-Vpr1 (left) and anti-SN (right) antibodies. FIG. 2B shows thatreplica blots, prepared from rVT7 infected HeLa cells transfected withpTM1, pTM-vpx2, pTM-vpx2SN and pTM-vpx2SN*, were probed with anti-Vpx2(left) and anti-SN (right) antibodies. Bound antibodies were detected byECL (Amersham) methods as described by the manufacturer.

FIG. 3 shows the incorporation of Vpr1- and Vpx2-SN and SN* fusionproteins into virus-like particles (VLP). FIG. 3A transfection of T7expressing (rVT7 infected) HeLa cells with pTM-vpr1, pTM-vpr1SN, andpTM-vpr1 SN* alone and in combination with pTM-gag1. pTM1 was alsotransfected for control. Culture supernatant were collected twenty-fourhours after transfection, clarified by centrifugation (1000×g, 10 min.)and ultracentrifuged (125,000×g, 2 hrs.) over cushions of 20% sucrose.Pellets (VLPs, middle and bottom panels) and cells (top panel) weresolubilized in loading buffer and examined by immunoblot analysis usinganti-Vpr1 (top and middle) and anti-Gag (bottom) antibodies as probes.FIG. 3B transfection of T7 expressing HeLa cells pTM-vpx2, pTM-vpx2SNand pTM-vpx2SN* alone and in combination with pTM-gag2. Pellets (VLPs,middle and bottom panels) and cells (top panel) were lysed, proteinswere separated by SDS-PAGE and electroblotted to nitrocellulose asdescribed above. Replica blots were probed with anti-Vpx2 (top andmiddle panels) and anti-Gag (bottom panel) antibodies. Bound antibodieswere detected using ECL methods.

FIG. 4 shows that virus-specific signals mediate incorporation of Vpr-and Vpx-SN into VLPs. FIG. 4A shows that HIV-1 Gag mediates packaging ofVpr1SN. rVT7 infected (T7 expressing) HeLa cells were transfected withpTM-vpr1 SN alone and in combination with pTM-gag2 and pTM-gag1. Pellets(VLPs, middle and bottom panels) and cells (top panel) were prepared 24hours after transfection and examined by immunoblot analysis usinganti-Vpr1 (top and middle) and anti-Gag (bottom) antibodies for probes.(B) HIV-2 Gag mediates packaging of Vpx2SN. T7 expressing HeLa cellswere transfected with pTM-vpx2SN alone and in combination with pTM-gag1and pTM-gag2. Pellets (VLPs, middle and bottom panels) and cells (toppanel) were prepared 24 hours after transfection and examined byimmunoblot analysis using anti-Vpx2 (top and middle) and anti-Gag(bottom) antibodies for probes.

FIG. 5 shows a competition analysis of Vpr1SN and Vpx2SN forincorporation into VLPs. FIG. 5A shows transfection of T7 expressingHeLa cells with different amounts of pTM-vpr1 (2.5, 5 and 10 μg) andpTM-vpr1 SN (2.5, 5 and 10 μg), either individually or together incombination with pTMgag1 (10 μg). FIG. 5B shows that HeLa cells weretransfected with different amounts of pTM-vpx2 (2.5, 5 and 10 μg) andpTM-vpx2SN (2.5, 5 and 10 μg), either individually or together withpTM-gag2 (10 μg). Twenty hours after transfection, particles wereconcentrated by ultracentrifugation through sucrose cushions andanalyzed by immunoblotting using anti-Vpr1 (A) or anti-Vpx2 (B)antibodies.

FIG. 6 shows the nuclease activity of VLP-associated Vpr1SN and Vpx2SNproteins. Virus-like particles were concentrated from culturesupernatants of T7 expressing HeLa cells cotransfected withpTM-gag1/pTM-vpr1SN, pTM-gag1/pTM-vpr1 SN*, pTM-gag2/pTM-vpx2SN andpTMgag2/pTM-vpx2SN* by ultracentrifugation (125,000×g, 2 hrs) through20% cushions of sucrose. Pellets containing Vpr1-SN and SN* (B) andVpx2-SN and SN* (C) were resuspended in PBS. Tenfold dilutions were madein nuclease reaction cocktail buffer (100 mM Tris-HCl pH 8.8, 10 mMCaCl₂, 0.1% NP40) and boiled for 1 minute. 5 μl of each dilution wasadded to 14 ul of reaction cocktail buffer containing 500 ng of lambdaphage DNA (HindIII fragments) and incubated at 37° C. for 2 hours.Reaction products were electrophoresed on 0.8% agarose gels and DNA wasvisualized by ethidium bromide staining. Standards (A) were prepared bydilution of purified staphylococcal nuclease (provided by A. Mildvan)into cocktail buffer and assayed.

FIG. 7 shows the incorporation of Vpx2SN into HIV-2 by transcomplementation. FIG. 7A shows the construction of the pLR2P-vpx2SN/SN*expression plasmids. To facilitate efficient expression of HIV genes,the HIV-2 LTR and RRE were engineered into the polylinker of pTZ19U,generating pLR2P. The organization of these elements within the pTZ19Upolylinker is illustrated. NcoI/XhoI vpx2SN and vpx2SN* (vpxSN/SN*)containing DNA fragments were ligated into pLR2P, generatingpLR2P-vpx2SN and pLR2P-vpx2SN* (pLR2P-vpxSN/SN*). FIG. 7B shows theassociation of Vpx2SN with HIV-2 virions. Monolayer cultures of HLtatcells were transfected with HIV-2_(ST) proviral DNA (pSXB1) andcotransfected with pSXB1/pTM-vpx2SN and pSXB1/pTM-vpx2SN*. Extracellularvirus was concentrated from culture supernatants forty-eight hours aftertransfection by ultracentrifugation (125,000×g, 2 hrs.) through cushionsof 20% sucrose. Duplicate Western blots of viral pellets were preparedand probed independently with anti-Vpx2 (left) anti-SN (middle) andanti-Gag (right) antibodies. FIG. 7C shows a sucrose gradient analysis.Pellets of supernatant-virus prepared from pSXB1/pTM-vpx2SNcotransfected HLtat cells were resuspended in PBS, layered over a 20-60%linear gradient of sucrose and centrifuged for 18 hours at 125,000×g.Fractions (0.5 ml) were collected from the bottom of the tube, diluted1:3 in PBS, reprecipitated and solubilized in electrophoresis buffer forimmunoblot analysis. Replica blots were probed with anti-SN (top) andanti-Gag (bottom) antibodies. Fraction 1 represents the first collectionfrom the bottom of the gradient and fraction 19 represents the lastcollection. Only alternate fractions are shown, except at the peak ofprotein detection. FIG. 7D shows the incorporation of Vpx2SN intoHIV-2_(7312A) Vpr and Vpx competent virus. Virus concentrated fromsupernatants of HLtat cells transfected with HIV-2_(7312A) proviral DNA(pJK) or cotransfected with pJK/pLR2P-vpx2SN or pJK/pLR2P-vpx2SN* wasprepared for immunoblot analysis as described above. Included forcontrol were virions derived by pSXB1/pLR2P-vpx2SN* cotransfection.Duplicate blots were probed with anti-Vpx (left) and anti-Gag (right)antibodies.

FIG. 8 shows the incorporation of Vpr1 SN into HIV-1 virions by transcomplementation. Culture supernatant virus from HLtat cells transfectedwith pNL4-3 (HIV-1) and pNL4-3R (HIV-1 vpr mutant) or cotransfected withpNL4-3/pLR2P-vpr1SN and pNL4-3R/pLR2P-vpr1SN was prepared for immunoblotanalysis as described above. Blots were probed with anti-SN (FIG. 8A),anti-Vpr1 (FIG. 8B) and anti-Gag (FIG. 8C) antibodies.

FIG. 9 shows the inhibition of Vpr1/Vpx2-SN processing by an HIVprotease inhibitor. HIV-1 (pSG3) and HIV-2 (pSXB1) proviral DNAs werecotransfected separately into replica cultures of HLtat cells withpLR2P-vpr1SN and pLR2P-vpx2SN, respectively. One culture of eachtransfection contained medium supplemented with 1 μM of the HIV proteaseinhibitor L-699-502. Virions were concentrated from culture supernatantsby ultracentrifugation through cushions of 20% sucrose and examined byimmunoblot analysis using anti-Gag (FIG. 9A) and anti-SN (FIG. 9B)antibodies.

FIG. 10 shows the incorporation of enzymatically active Vpr1- andVpx2-CAT fusion proteins into HIV virions. FIG. 10A shows anillustration of the fusion junctions of the pLR2P-vpr1CAT andpLR2P-vpx2CAT expression plasmids (SEQ ID NOS:15, 16, 17 and 18). PCRamplified BamHI/XhoI DNA fragments containing CAT were ligated into Bg1II/XhoI cut pLR2P-vpr1SN and pLR2P-vpx2SAN, replacing SN (see FIG. 1).This construction introduced two additional amino acid residues (Asp andLeu, above blackened bar) between the vpr1/vpx2CAT coding regions. FIG.10B shows the incorporation of Vpr1CAT into HIV-1 virions. Virusproduced from HLtat cells transfected with pNL4-3 (HIV-1) and pNL4-3R(HIV-1-R), or cotransfected with pNL4-3/pLR2P-vpr1 CAT andpNL4-3R/pLR2P-vpr1 CAT was prepared as described above and examined byimmunoblot analysis. Replica blots were probed with anti-Vpr1 (left) andanti-Gag (right) antibodies. FIG. 10C shows the incorporation of Vpx2CATinto HIV-2 virions. Virus produced from HLtat cells transfected withpSXB1 (HIV-2) or cotransfected with pSXB 1/pLR2P-vpx2CAT was prepared asdescribed above and examined by immunoblot analysis. Replica blots wereprobed with anti-Vpx2 (left) and anti-Gag (right) antibodies. FIG. 10Dshows that virion incorporated Vpr1- and Vpx2-CAT fusion proteinspossess enzymatic activity. Viruses pelleted from HLtat cellstransfected with pSXB1 (HIV-2) or cotransfected with pSXBI/pLR2P-vpx2CATand pNL4-3/pLR2P-vpr1 CAT were lysed and analyzed for CAT activity.HIV-2 was included as a negative control.

FIG. 11 shows virion association of enzymatically active CAT and SNfusion proteins. FIG. 11A shows that HIV-2 virions collected from theculture supernatant of HLtat cells cotransfected with pSXB1 andpLR2P-vpx2 were sedimented in linear gradients of 20-60% sucrose. 0.7 mlfractions were collected and analyzed by immunoblot analysis using Gagmonoclonal antibodies as a probe. FIG. 11B shows CAT enzyme activity wasdetermined in each fraction by standard methods. The positions ofnonacetylated [¹⁴C]chloramphenicol (Cm) and acetylated chloramphenicol(Ac—Cm) are indicated. FIG. 11C shows HIV-1 virions derived from HLtatcells cotransfected with pSG3 and pLR2P-vpr1SN and cultured in thepresence of L-689,502 were sedimented in linear gradients of 20-60%sucrose. Fractions were collected and analyzed for virus content byimmunoblot analysis using Gag monoclonal antibodies. FIG. 11D shows thatSN activity was determined in each fraction as described in FIG. 6.

FIG. 12 shows the HIV-1 genome, the construction of pΔ8.2, pCMV-VSV-G,pHR-CMV-β-gal, pCR-gag-pro, pLR2P-vpr-RT-IN, pCMV-VSV-G andpHR-CMV-β-gal plasmids. FIG. 12A shows an illustration of the HIV-1genome. FIG. 12B shows the lentivirus vector plasmid expression system.FIG. 12C shows the illustration of a trans-lentiviral vector expressionsystem, where RT and IN are contiguous as Vpr fusion partners.

FIG. 13 shows positive gene transduction with a trans-lentiviral vectorof the instant invention as determined by fluorescence microscopy.

FIG. 14 shows positive gene transduction with a lentiviral vector as acontrol as determined by fluorescence microscopy.

FIG. 15 shows the construction of a pHR-CFTR trans-lentiviral vector ofthe present invention.

FIG. 16 shows the expression of CFTR on HeLa cells using thetrans-lentiviral vector, and the lentiviral vector as a control.Transduced cells were probed with polyclonal antibodies inimmunofluorescence microscopy.

FIG. 17 shows the expression of CFTR on HeLa cells using monoclonalantibodies in immunofluorescence microscopy.

FIG. 18 shows the restoration of CFTR function in trans-lentiviraltransduced HeLa cells as measured by a halide sensitive fluorophore.

FIGS. 19A and B show the presence in progeny virions of RT in transwithout Vpr-dependent incorporation.

FIG. 20 shows that both Vpr-RT and RT support vector transduction whenprovided in trans.

FIG. 21 shows component constructs of a trans-retroviral vectoraccording to the present invention. FIG. 21 A shows a pCMV, Gag-Propackaging plasmid. FIG. 21 B shows a pCMV, GagNC-RT-IN trans-enzymeexpression plasmid. FIG. 21C shows a vector plasmid. FIG. 21D shows anenvelope plasmid construct operative in the present invention.

FIG. 22 shows component constructs of a retroviral vector according tothe present invention. FIG. 22A shows a pCMV, Gag-Pro-RT-IN retroviralpackaging plasmid. FIGS. 22B and C are the vector plasmid and envelopeplasmids of FIGS. 21C and D, respectively.

FIG. 23 shows component constructs of a lentiviral vector according tothe present invention. FIG. 23A shows a pTRE, Gag-Pro-RT-IN packagingplasmid. FIG. 23B shows a pHR-CTS, CMV, GFP, WPRE lentiviral vectorplasmid. FIG. 23C is the envelope plasmid at FIG. 21D.

FIGS. 24(A)-(C) show representative trans-lentiviral trans-enzymeplasmids according to the present invention indicating Pro cleavagesites and zinc finger locations.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term “fusion protein” refers to either the entirenative protein amino acid sequence of Vpx (of any HIV-2 and SW) or Vpr(of any HIV-1 and SIV) or retroviral Gag or any subfraction of theirsequences that have been joined through recombinant DNA technology andare capable of association with either native HIV/SIV virions or aretrovirus-like particle.

As used herein, the term “virion” refers to HIV-1, HIV-2 and SIV virusparticles.

As used herein, the term “retrovirus-like particle” refers to anycomposition of HIV-1, HIV-2, SIV or retrovirus proteins other than whichexists naturally in naturally infected hosts that are capable ofassembly and release from either natural or immortalized cells thatexpress these proteins.

As used herein, the term “variant” refers to a polypeptide or nucleotidesequence having at least 30% sequence identity with the native sequenceincluding fragments thereof as calculated by Fast DB as per “CurrentMethods in Sequence Comparison and Analysis,” Macromolecule Sequencingand Synthesis, Selected Methods and Applications, pp. 127-149.

As used herein, the term “transfect” refers to the introduction ofnucleic acids (either DNA or RNA) into eukaryotic or prokaryotic cellsor organisms.

As used herein, the term “gene transduction element” refers to theminimal required genetic information to transduce a cell with a gene.

As used herein, the term “virus-inhibitory protein” refers to anysequence of amino acids that have been fused with Vpx or Vpr or Gagsequences that may alter in any way the ability of a retrovirus tomultiply and spread in either individual cells (prokaryotic andeukaryotic) or in higher organisms. Such inhibitory molecules mayinclude: HIV/SIV proteins or sequences, including those that may possessenzymatic activity (examples may include the HIV/SIV protease,integrase, reverse transcriptase, Vif and Nef proteins) HIV/SIV proteinsor proteins/peptide sequences that have been modified by geneticengineering technologies in order to alter in any way their normalfunction or enzymatic activity and/or specificity (examples may includemutations of the HIV/SIV protease, integrase, reverse transcriptase, Vifand Nef proteins), or any other non-viral protein that, when expressedas a fusion protein with Vpr or Vpx or Gag, alter virus multiplicationand spread in vitro or in vivo.

In the present invention, the HIV Vpr and Vpx proteins were packagedinto virions through virus type-specific interactions with the Gagpolyprotein precursor. HIV-1 Vpr (Vpr1) and HIV-2 Vpx (Vpx2) areutilized to target foreign proteins to the HIV particle as their openreading frames were fused in-frame with genes encoding the bacterialstaphylococcal nuclease (SN), an enzymatically inactive mutant ofSN(SN*), and the chloramphenicol acetyl transferase (CAT). Transientexpression in a T7-based vaccinia virus system demonstrated thesynthesis of appropriately sized Vpr1SN/SN* and Vpx2SN/SN* fusionproteins which, when co-expressed with their cognate p55^(Gag) protein,were efficiently incorporated into virus-like particles (VLPs).Packaging of the fusion proteins was dependent on virus type-specificdeterminants, as previously seen with wild-type Vpr and Vpx proteins.Particle associated Vpr1SN and Vpx2SN fusion proteins were enzymaticallyactive as determined by in vitro digestion of lambda phage DNA. Todemonstrate that functional Vpr1 and Vpx2 fusion proteins were targetedto HIV particles, the gene-fusions were cloned into an HIV-2 LTR/RREregulated expression vector and co-transfected with wild-type HIV-1 andHIV-2 proviruses. Western blot analysis of sucrose gradient purifiedvirions revealed that both Vpr1 and Vpx2 fusion proteins wereefficiently packaged regardless of whether SN, SN* or CAT were used as Cterminal fusion partners. Moreover, the fusion proteins remainedenzymatically active and were packaged in the presence of wild-type Vprand Vpx proteins. Interestingly, virions also contained smaller sizedproteins that reacted with antibodies specific for the accessoryproteins as well as SN and CAT fusion partners. Since similar proteinswere absent from Gag-derived VLPs as well as in virions propagated inthe presence of an HIV protease inhibitor, they must represent cleavageproducts produced by the viral protease. Taken together, these resultsdemonstrate that Vpr and Vpx can be used to target functional proteins,including potentially deleterious enzymes, to the HIV/SIV particle.These properties are useful for the development of novel antiviralstrategies.

In the present invention, a gene cassette is coupled to a retrovirus Gagvariant within a trans-enzyme plasmid to induce fusion proteinexpression of the gene. Through selection of the gene and modificationof the Gag nucleotide sequence, the vectors of the present invention areoperative as antiviral therapeutics and/or gene delivery vectors whentransfected into host cells in conjunction with genes or variantsthereof coding packaging, vector and envelope polypeptides. While thepresent invention is detailed herein with plasmids each encodingdifferent vector functions, it is appreciated that such functions arereadily combined into a lesser number of plasmids including one, two andthree plasmids which are cotransfected into a host cell. Preferably, amultiple plasmid gene delivery system is utilized according to thepresent invention.

A Gag based trans-lentiviral vector was produced by transfecting 293Tcells with the pCMV-gag-pro (packaging plasmid), a differenttrans-enzyme plasmid based on Gag, the pPCMV-eGFP (transfer vector), andthe pMD-G (env plasmid). The Gag based trans-lentiviral vector of thepresent invention demonstrates that the Gag precursor protein is able todeliver function fusion proteins to a host cell. The fusion proteinsillustratively including RT, IN, RT-IN, GFP, CAT, CFTR and the like. Asa control, trans-lentiviral vector based Vpr was produced bytransfecting 293T cells with the pCMV-gag-pro (packaging plasmid), thepLR2P-Vpr-RTIN (trans-enzyme plasmid), the pPCMV-eGFP (transfer vector),and the pMD-G (env plasmid) (Wu et al. (1997) EMBO Journal16:5113-5122). Using fluorescence microscopy to monitor GFP expression,the infectivity of the trans-lentiviral vector particles was monitoredon monolayer cultures of HeLa cells. As shown in Table 1, the titer ofthe trans-lentiviral vector based on Gag ranged from 0.4 to 4×10⁵/ml,while that of the trans-lentiviral vector based on Vpr ranged from 5 to9×10⁵/ml. The Gag precursor protein according to the present inventionis capable of delivering functional proteins into the vector particles.Reproducibly, the titer of the trans-lentiviral vector based Gag wasapproximately 2-5 fold less than that of the trans-lentiviral vectorbased Vpr for RT-IN.

TABLE 1 Titers of Trans-Lentiviral GagRTIN Vectors Con- Viral Controlstructs Trans-Lentiviral Delivery Vectors Titer Viral Titer* A pTRE,GagNC-RT-IN, RRE-1  3.5 × 10⁴   5 × 10⁵ B pPLR2P-GagNC(1ZF)-RT-IN, 3.75× 10⁵  6.5 × 10⁵ RRE-2** C pLR2P-GagCA-RT-IN, RRE-2*** 1.25 × 10⁵ 8.75 ×10⁵ D pLR2P-GagNC-RT-IN, RRE-2  2.5 × 10⁵   5 × 10⁵ EpLR2P-GagNC(ΔPC)-RT-IN, 1.25 × 10⁵   5 × 10⁵ RRE-2**** *pLR2P-Vpr-RT-INplasmid was used as positive control. **The 3′ Zinc Finger domain wasdeleted in the NC domain of this construct. ***The whole NC domain wasdeleted in this construct. ***Only the Pro-RT protease cleavage siteexists between the Gag and RT domains of this construct.

The ability of trans-RT-IN to support virus infectivity of thelentivirus particles (virions derived from RT-IN minus proviral DNA ofHIV-1) or a lentivirus-based vector, indicates that trans-RT-IN fusionprotein is readily substituted for cis acting RT-IN. To determinewhether the trans-RT-IN (derived from the Gag-RT-IN fusion protein) of asimple retrovirus, like the lentivirus, also cis acting RT-TN derivedfrom the native Gag-Pol structure (GAG-PR-RT-IN) is replaced bytrans-RT-IN derived from Gag-RT and Gag-IN or a triple fusion ofGag-RT-IN in a retrovirus such as a lentivirus. Thus, a trans-retroviralvector based Gag was produced by transfecting 293T cells with the 5 μgof packaging construct (pCMV-ATG/gag-pro), 2 μg of the trans-enzymeplasmid (pCMV-ATG/gag-RT-IN), 5 μg of the transfer vector(pRTCMV-eGFP-WPRE) and the pMD-G (env plasmid). As a control, theretrovirus vector was produced by transfecting 293T cells with thepCMV-ATG/gag-pol (packaging plasmid), 5 μg of the transfer vector(pRTCMV-eGFP-WPRE) and the pMD-G (env plasmid). Using fluorescencemicroscopy to monitor GFP expression, the infectivity of thetrans-lentiviral vector particles was monitored on monolayer cultures ofHeLa cells. As shown in Table 2, the titer of the trans-retrovirusvector ranged from 0.6 to 1.8×10⁷/ml. Retrovirus vector titers rangedfrom 0.8 to 2.5×10⁷/ml. This result demonstrates that the simple Gagprecursor protein of a retrovirus also can deliver the functionalproteins into a vector particle in trans. Thus, according to the presentinvention gene delivery to a host cell occurs with a Gag precursor geneas a fusion partner to a protein of interest, thereby making a varietyof retroviruses operative as gene delivery vector systems.

TABLE 2 Titers of the Trans-Retroviral and Retroviral VectorsTrans-enzyme Packaging Envelope Plasmid Plasmid Vector Plasmid PlasmidViral Titer NA* pCMV, Gag-Pro-RT-IN pRT-CMV, GFP, WPRE pMD-G 7.18 × 10⁶NT** pCMV, Gag-Pro pRT-CMV, GFP, WPRE pMD-G   6 × 10³ pCMV, GagNC- NT**pRT-CMV, GFP, WPRE pMD-G 0 RT-IN pCMV, GagNC- pCMV, Gag-Pro pRT-CMV,GFP,WPRE pMD-G 1.78 × 10⁷ RT-IN *NA not applicable **NT Not transfected

Gag fusions are operative here from retroviruses and lentivirusesincluding Moloney Leukemia Virus (MLV), Abelson murine leukemia virus,AKR (endogenous) murine leukemia virus, Avian carcinoma, Mill Hill virus2, Avian leukosis virus-RSA, Avian myeloblastosis virus, Avianmyelocytomatosis virus 29, Bovine syncytial virus, Caprine arthritisencephalitis virus, Chick syncytial virus, Equine infectious anemiavirus, Feline leukemia virus, Feline syncytial virus,Finkel-Biskis-Jinkins murine sarcoma virus, Friend murine leukemiavirus, Fujinami sarcoma virus, Gardner-Arnstein feline sarcoma virus,Gibbon ape leukemia virus, Guinea pig type C oncovirus, Hardy-Zuckermanfeline sarcoma virus, Harvey murine sarcoma virus, Human foamy virus,Human spumavirus, Human T-lymphotropic virus 1, Human T-lymphotropicvirus 2, Jaagsiekte virus, Kirsten murine sarcoma virus, Langur virus,Mason-Pfizer monkey virus, Moloney murine sarcoma virus, Mouse mammarytumor virus, Ovine pulmonary adenocarcinoma virus, Porcine type Concovirus, Reticuloendotheliosis virus, Rous sarcoma virus, Simian foamyvirus, Simian sarcoma virus, Simian T-lymphotropic virus, Simian type Dvirus 1, Snyder-Theilen feline sarcoma virus, Squirrel monkeyretrovirus, Trager duck spleen necrosis virus, UR2 sarcoma virus, Viperretrovirus, Visna/maedi virus, Woolly monkey sarcoma virus, and Y73sarcoma virus human-, simian-, feline-, and bovine immunodeficiencyviruses (HIV, SIV, FIV, BIV). While RT and IN fusions with Gag areoperative herein, it is appreciated that a variety of therapeutic anddiagnostic fusion proteins are similarly deliverable to a target cellaccording to the methodologies and vectors disclosed herein.

Gag-based trans-lentiviral vectors are disclosed based on non-primatelentiviruses and simple retroviruses encoding retrovirus Gag precursorproteins which have functions akin to those of primate lentiviral Vpr orVpx proteins. Contrary to the prior art, the present invention maintainsviral infectivity in non-dividing primary cells. The WPRE sequenceencoded within a vector of the present invention is necessary therefor.Infectivity of the vectors of the present invention is further enhancedthrough the use of a gene transfer vector containing thepost-transcriptional regulatory element of woodchuck hepatitis virus(WPRE). While the inclusion of a WPRE gene or gene fragment capable ofregulating post-transcription increases trans-lentiviral titer alone,the inclusion of additional PPT-CTS sequences creates a cumulativeenhancement in viral infectivity. WPRE has been shown to increaseluciferase or GFP production in similar virus-based vectors (Zufferey etal. (1999) J. Virol. 73:2886-2892. Alternatively, the central terminatorsequence (CTS) and central polypurine tract (PPT) are introduced intothe gene transfer vector to independently increase titer, as detailed inU.S. Provisional Application 60/164,626 filed Nov. 10, 1999. PPT and CTShave been implicated in HIV-1 reverse transcription. Charneau et al.(1994) 1 Mol. Biol. 241:651-662. It is appreciated that the othercontrol sequences capable of stabilizing messenger RNA and therebyfacilitating protein expression are operative in place of WPRE, PPT, andCTS within the present invention.

The present invention provides for a delivery of a trans-protein or geneto a viral vector through coupling to either a viral protein or genedelivery, respectively; wherein the viral protein is Vpr or Vpx or Gagand the gene encodes either Vpr or Vpx or Gag. Certain truncationvariants of these trans-proteins or genes perform the regulatory orenzymatic functions of the full sequence protein or gene. For example,the nucleic acid sequences coding for Protease, Integrase, ReverseTranscriptase, Vif, Nef, Gag, and CFTR can be altered by substitutions,additions, deletions or multimeric expression that provide forfunctionally equivalent proteins or genes. Due to the degeneracy ofnucleic acid coding sequences, other sequences which encodesubstantially the same amino acid sequences as those of the naturallyoccurring proteins may be used in the practice of the present invention.These include, but are not limited to, nucleic acid sequences comprisingall or portions of the nucleic acid sequences encoding the aboveproteins, which are altered by the substitution of different codons thatencode a functionally equivalent amino acid residue within the sequence,thus producing a silent change. For example, one or more amino acidresidues within a sequence can be substituted by another amino acid of asimilar polarity which acts as a functional equivalent, resulting in asilent alteration. Substitutes for an amino acid within the sequence maybe selected from other members of the class to which the amino acidbelongs. For example, the nonpolar (hydrophobic) amino acids includealanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophanand methionine. The polar neutral amino acids include glycine, serine,threonine, cysteine, tyrosine, asparagine, and glutamine. The positivelycharged (basic) amino acids include arginine, lysine and histidine. Thenegatively charged (acidic) amino acids include aspartic acid andglutamic acid. Also included within the scope of the present inventionare proteins or fragments or derivatives thereof which aredifferentially modified during or after translation, e.g., byglycosolation, protolytic cleavage, linkage to an antibody molecule orother cellular ligands, etc. In addition, the recombinant ligandencoding nucleic acid sequences of the present invention may beengineered so as to modify processing or expression of a ligand. Forexample, a signal sequence may be inserted upstream of a ligand encodingsequence to permit secretion of the ligand and thereby facilitateapoptosis.

Additionally, a ligand encoding nucleic acid sequence can be mutated invitro or in vivo to create and/or destroy translation, initiation,and/or termination sequences or to create variations in coding regionsand/or form new restriction endonuclease sites or destroy pre-existingones, to facilitate further in vitro modification. Any technique formutagenesis known in the art can be used, including but not limited toin vitro site directed mutagenesis, J. Biol. Chem. 253:6551, use of Tablinkers (Pharmacea), etc.

The following examples are given for the purpose of illustrating variousembodiments of the invention and are not meant to limit the presentinvention in any fashion.

EXPERIMENTAL Example 1 Cells and Viruses

HeLa, HeLa-tat (HLtat), 293T and CV-1 cells were maintained inDulbecco's Modified Eagle's Medium supplemented with 10% fetal bovineserum (FBS), 100 U penicillin and 0.1 mg/ml streptomycin. HLtat cellsconstitutively express the first exon of HIV-1 tat and were provided byDrs. B. Felber and G. Pavlakis. A recombinant vaccinia virus (rVT7)containing the bacteriophage T7 RNA polymerase gene was used tofacilitate expression of viral genes placed under the control of a T7promoter. Stocks of rVT7 were prepared and titrated in CV-1 cells asdescribed previously by Wu et al. (1992) J Virol. 66:7104-7112.HIV-1_(YU2), HIV-1 pNL 4-3-R and pNL 4-3, HIV-1_(HXB2), H1V-2_(ST), andHIV-2_(7312A) proviral clones were used for the construction ofrecombinant expression plasmids and the generation of transfectionderived viruses.

Example 2 Antibodies

To generate HIV-1 Vpr specific antibodies, the HIV-1_(YU-2) vpr openreading frame was amplified by polymerase chain reaction (PCR) usingprimers (sense: 5′-GCCACCTTTGTCGACTGTTAAAAAACT-3′ (SEQ ID NO:1) andantisense: 5′-GTCCTAGGCAAGCTTCCTGGATGC-3′ (SEQ ID NO:2)) containing Sa1Iand HindIII sites and ligated into the prokaryotic expression vector,pGEX, generating pGEX-vpr1. This construct allowed expression of Vpr1 asa C terminal fusion protein and Glutathione S-Transferase (GST), thusallowing protein purification using affinity chromatography. E. coli(DH5d) were transformed with pGEX-vpr1 and protein expression wasinduced with isopropyl (β-D thiogalactopyranoside (IPTG)). Expression ofthe GST-Vpr1 fusion protein was confirmed by SDS-PAGE. Soluble GST-Vpr1protein was purified and Vpr1 was released by thrombin cleavage usingpreviously described procedures of Smith et al. (1988) Gene 67:31-40.New Zealand White rabbits were immunized with 0.4 mg of purified Vpr1protein emulsified 1:1 in Freunds complete adjuvant, boosted three timesat two week intervals with 0.25 mg of Vpr1 mixed 1:1 in Freunds'incomplete adjuvant and bled eight and ten weeks after the firstimmunization to collect antisera. Additional antibodies used includedmonoclonal antibodies to HIV-1 Gag (ACT1, and HIV-2 Gag (6D2.6),polyclonal rabbit antibodies raised against the HIV-2 Vpx protein andanti-SN antiserum raised against purified bacterially expressed SNprotein.

Example 3 Construction of T7-Based Expression Plasmids

A DNA fragment encompassing ^(HIV-1)HXB2D^(gag) (nucleotides 335-1837)was amplified by PCR using primers (sense: 5′-AAGGAGAGCCATGGGTGCGAGAGCG-3′ (SEQ ID NO:3) and anti-sense: 5-GGGGATCC CTTTATTGTGACGAGGGG-3′ (SEQ ID NO:4)) containing NcoI and BamHI restriction sites(underlined). The PCR product was digested with NcoI and BamHI, purifiedand ligated into the polylinker of the pTM1 vector, generating pTM-gag1.Similarly, a DNA fragment containing the Gag coding region ofHIV-2_(ST), (nucleotides 547-2113) was amplified by PCR using sense andanti-sense primers 5′-ATTGTGGGCCATGGGCGCGAGAAAC-3′ (SEQ ID NO:5) and5′-GGGGGG CCCCTACTGGTCTTTTCC-3 (SEQ ID NO:6), respectively. The reactionproduct was cut with NcoI and SmaI (underlined), purified and ligatedinto the polylinker of pTM1, generating pTM-gag2.

For expression of Vpr1 under the control of the T7 promoter, a DNAfragment containing the HIV-1_(YU2) vpr coding region (nucleotides5107-5400) was amplified by PCR using primers (sense: 5′-GAAGATCTACCATGGAAGCCCCAGAAGA-3′ (SEQ ID NO:7) and anti-sense: 5′-CGCGGATCCGTTAACATCTACTGGCTCCATTTCTTGCTC-3′ (SEQ ID NO:8)) containing NcoI and HpaI/BamHIsites, respectively (underlined). The reaction product was cut with NcoIand BamHI and ligated into pTM1, generating a pTM-vpr1 (FIG. 12A). Inorder to fuse SN and SN* in-frame with vpr1, their coding regions wereexcised from pGN1561.1 and pGN1709.3, respectively and through a seriesof subcloning steps, ligated into the SmaI/XhoI sites of pTM-vpr1,generating pTM-vpr1SN and pTM-vpr1SN*. This approach changed thetranslational stop codon of Vpr1 to a Trp codon and the C terminal Serresidue to a Cys. The resulting junctions between vpr1 and SN/SN* aredepicted in FIG. 12C.

For expression of Vpx2 under T7 control, a DNA fragment containing theHIV-2_(ST) vpx coding sequence (nucleotides 5343-5691) was amplified byPCR using primers (sense: 5′-GTGCAACACCATGGCAGGCCCCAGA-3′ (SEQ ID NO: 9)and anti-sense: 5′-TGCACTGCAGGAAGATCTTAGACCTGGAGGGGGAG GAGG-3′ (SEQ IDNO: 10)) containing NcoI and Bg1 II sites, respectively (underlined).After cleavage with BgLII and Klenow fill-in, the PCR product wascleaved with NcoI, purified and ligated into the NcoI and SmaI sites ofpTM1, generating pTM-vpx2 (FIG. 12B). To construct in-frame fusions withvpx2, BamHI/XhoI, SN- and SN*-containing DNA fragments were excised frompTM-vpr1SN and pTM-vpr1SN* and ligated into pTM-vpx2, generatingpTM-vpx2SN and pTM-vpx2SN*, respectively. This approach introduced oneamino acid substitution at the C terminus of Vpx (Val to Arg), changedthe translational stop codon of vpx to Ser and left five amino acidsresidues of the pTM1 plasmid polylinker. The resulting junctions betweenvpx2 and SN/SN* are depicted in FIG. 1D.

Example 4 Construction of HIV LTR-Based Vpr or Vpx Expression Plasmids

For efficient expression of Vpr and Vpx fusion proteins in the presenceof HIV, a eukaryotic expression vector (termed pLR2P) was constructedwhich contains both an HIV-2 LTR (HIV-2_(ST), coordinates −544 to 466)and an HIV-2 RRE (HIV-2_(ROD), coordinates 7320 to 7972) element (FIG.7A). These HIV-2 LTR and RRE elements were chosen because they respondto both HIV-1 and HIV-2 Tat and Rev proteins. The vpr1, vpr1SN,vpx2 andvpx2SN coding regions were excised from their respective pTM expressionplasmids (see FIG. 1) with NcoI and XhoI restriction enzymes and ligatedinto pLR2P, generating pLR2P-vpr1, pLR2P-vpr1SN, pLR2P-vpx2 andpLR2P-vpx2SN, respectively (FIG. 7A). For construction and expression ofvpr- and vpx-CAT gene fusions, the SN containing regions (BamHI/XhoIfragments) of pLR2P-vpr1SN and pLR2P-vpx2SN were removed and substitutedwith a PCR amplified Bgl II/XhoI DNA fragment containing CAT, generatingpLR2P-vpr1CAT and pLR2P-vpx2CAT, respectively (FIG. 9A).

Example 5 Construction of Lentiviral Plasmids Involving Gag Fusions

The pHRCMV-eGFP plasmid was derived by modifying pHRCMV-LacZ which hasbeen described (Naldini et al. (1996) Science 272:263-267). ThepHRCMV-eGFP plasmid was constructed by ligating a BamHI/XhoI DNAfragment containing eGFP (derived from pEGFP-C1; CLONTECH Laboratories,Palo Alto, Calif.) into the pHRCMV-lacz plasmid after removing lacz bydigestion with BamHI and XhoI. To construct the pPCMV-eGFP, a 150 bpsequence (with coordinates 4327-4483) and containing the central PPT andcentral terminal site (CTS) was amplified from the SG3 molecular cloneby PCR and ligated into pHRCMV-eGFP that was cut with ClaI. To constructthe Tet-inducible expression plasmids, 430 bps of TRE-inducible promoterderived from pTRE; CLONTECH Laboratories, Palo Alto, Calif., was cut byXhoI filled to blunt ends and BamHI. The CMV promoter of pcDNA3.1(+)plasmid (Invitrogen, CA) was replaced using SpeI (filled to blunt ends)and BamHI, generating pTRE-neo. The 6.7 kb fragment containing theHIV-based packaging components derived from pCMVgag-pol was cloned intopTRE-neo using EcoRI and XhoI, generating pTRE-gag-pol which containsfunctional vif, tat, rev, gag and pal genes. To construct the RT-INminus plasmid shown in FIG. 23A, the region (from 1975 to 5337) ofpTREgag-pol were substituted with an RT-IN containing BcII/Sa1I DNAfragment (from 1975 to 5337) of pSG3S-RT was ligated into the Bc1I andSa1I sites of the pTREgag-pol plasmid, generating pTREgag-pro. The RT-INsequence contained translational stop codons (TAA) at the first aminoacid position of the RT and IN coding regions and was under control ofCMV promoter. A 39-base pair internal deletion in the 4 sequence wasintroduced, the internal region (1357 bp) of envelope gene was deletedfrom 5827 to 7184, generating pCMVgag-pol. To construct the RT-IN minusplasmid, the region (from 1975 to 5337) of pCMVgag-pol was substitutedwith an RT-IN containing Bc1I/Sa1I DNA fragment (from 1975 to 5337) ofpSG3S-RT was ligated into the Bc1I and Sa1I sites of the pCMVgag-polplasmid, generating pCMVgag-pro shown in Table 2. The RT-IN sequencecontained translational stop colons (TAA) at the first amino acidposition of the RT and IN coding regions. To construct the series oftrans-enzyme plasmids shown in FIG. 24A-C and Table 2, differentfragments of HIV-1 gag genes were amplified by PCR and were cloned intopLR2Pvpr-RT-IN using Nco1 1 and Bg1 11, generating a series of gag-RTINfusion expression plasmids. pLR2gagNC-RTIN shown in Table 1 as constructD contained the Gag gene with the p6 portion deleted.pLR2PgagNC(1ZF)-RTIN shown in FIG. 24B and contains the Gag gene withthe second Zing finger of NC and p1-p6 fragment deleted. pLR2PgagCA-RTINshown as FIG. 24C and in Table 1 as construct C contains the Gag genewhich deleted the NC-p1-p6 fragment. pLR2PVprRTIN construction isdescribed in FIG. 12. pMD-G is constructed according to existingtechniques (Wu et al. (1997) EMBO Journal 16:5113-5122).

Example 6 Construction of Retroviral Plasmids Involving Gag Fusions

A RT-IN minus packaging construct was formed based on Moloney murineleukemia virus. pCMV-ATG/gag-pol was cut by SalI and filled with Klenow,generating pCMV-ATG/gag-pro, with the RT gene being mutated by thereading frame shift at amino acid position 366 as shown in FIG. 21A. Togenerate a trans-enzyme plasmid, the 312 bp fragment which contains theprotease region was deleted from the gag-pol of pCMV-ATG/gag-pol,generating pCMV-ATG/gag-RT-IN as shown in FIG. 21B. The GFP transfervector based on Moloney murine leukemia virus, GFP-WPRE which wasobtained from pPCMV-eGFP-WPRE (Finer et al. (1994) Blood 83:43-50) andcloned into pRTCMV using BamHI and ApoI, generating pRTCMV-eGFP-WPRE asshown in FIG. 21C.

Example 7 Preparation of Vector Stocks and Infection

Trans-lentiviral vector stocks were produced by transfecting the 5 μg ofpackaging construct (pTREgag-pol), the 2 μg of VSV-G construct (pMD-G),and 5 μg of the transfer vector (pPCMV-eGFP WPRE) and 1 μg of pTet-off(CLONTECH Laboratories, Palo Alto, Calif.) and different trans-enzymeplasmids into the subcontinent 293T cell by the calcium phosphateprecipitation method. Trans-retroviral vector stocks were produced bytransfecting the 5 μg of packaging construct (pCMV-ATG/gag-pro), the 2μg of VSV-G construct (pMD-G), and 5 μg of the transfer vector(pRTCMV-eGFP-WPRE) and 2 μg of the trans-enzyme plasmid(pCMV-ATG/gag-RT-IN). Approximately 1×10⁶ cells were seeded intosix-well plates 24 hr prior to transfection. The vector stocks wereharvested 60 hr posttransfection. Supernatants of the transfectedcultures were clarified by low speed centrifugation (1000 g, 10 min),and filtered through a 0.45-μg-pore-size filter, aliquoted andsubsequently frozen at −80° C. The target cells were infected in theDMEM-1% FBS containing 10 μg/ml of DEAETestron for 4 hr at 37° C. Themedium was subsequently replaced with fresh DMEM-10% FBS orpreconditional medium. To determine the titer of eGFP vector, thesupernatant stock of 1.0, 0.2, 0.04, and 0.008 μl were used to infectthe culture of HeLa cell. 2-3 days later, positive (green) cell colonieswere counted using a fluorescence microscope.

Example 8 Transfections

Transfections of proviral clones were performed in HLtat cells usingcalcium phosphate DNA precipitation methods as described by themanufacturer (Strategene). T7-based (pTM1) expression constructs weretransfected using Lipofectin (BioRad) into rVT7 infected HeLa cells asdescribed previously by Wu et al. (1994) J. Virol. 68:6161-6169. Thesemethods were those recommended by the manufacturer of the Lipofectinreagent.

Example 9 Western Immunoblot Analysis

Virions and virus-like particles (VLPs) were concentrated from thesupernatants of transfected or infected cells by ultracentrifugationthrough 20% cushions of sucrose (125,000×g, 2 hrs., 4° C.). Pellets andinfected/transfected cells were solubilized in loading buffer (62.5 mMTris-HCl (pH 6.8) 0.2% sodium dodecyl sulfate (SDS), 5%2-mercaptoethanol, 10% glycerol), loaded and separated on 12.5%polyacrylamide gels containing SDS. Following electrophoresis, proteinswere transferred to nitrocellulose (0.2 μm; Schleicher 34 & Schnell) byelectroblotting, incubated for one hour at room temperature in blockingbuffer (5% nonfat dry milk in phosphate buffered saline [PBS]) and thenfor two hours with the appropriate antibodies diluted in blockingbuffer. Protein bound antibodies were detected with HRP-conjugatedspecific secondary antibodies using ECL methods according to themanufacturer's instructions (Amersham).

Example 10 SN Nuclease Activity Assay

Cells and viral pellets were resuspended in nuclease lysis buffer (40 mMTris-HCl, pH 6.8, 100 mM NaCl, 0.1% SDS, 1% Triton X-100) and clarifiedby low speed centrifugation (1000×g, 10 min.). Tenfold dilutions weremade in nuclease reaction cocktail buffer (100 mM Tris-HCl, pH 8.8, 10mM CaCl₂, 0.1% NP40) and boiled for 1 minute. 5 μl of each dilution wasadded to 14 μl of reaction cocktail buffer containing 500 ng of lambdaphage DNA (HindIII fragments) and incubated at 37° C. for 2 hours.Reaction products were electrophoresed on 0.8% agarose gels and DNA wasvisualized by ethidium bromide staining.

Example 11 Expression of Vpr1- and Vpx2-SN and SN* Fusion Proteins inMammalian Cells

Expression of Vpr1- and Vpx2-SN/SN* fusion proteins in mammalian cellswas assessed using the recombinant vaccinia virus-T7 system (rVT7). HeLacells were grown to 75-80% confluency and transfected with therecombinant plasmids pTM-vpr, pTM-vpx, pTM-vpr1 SN/SN*, andpTMvpx2SN/SN* (FIG. 1). Twenty-four hours after transfection, cells werewashed twice with PBS and lysed. Soluble proteins were separated bySDS-PAGE and subjected to immunoblot blot analysis. The results areshown in FIG. 2. Transfection of pTM-vpr1SN and pTM-vpr1SN* resulted inthe expression of a 34 kDa fusion protein that was detectable using bothanti-Vpr and anti-SN antibodies (A). Similarly, transfection ofpTM-vpx2SN and pTM-vpx2SN* resulted in the expression of a 35 kDa fusionprotein which was detected using anti-Vpx and anti-SN antibodies (B).Both fusion proteins were found to migrate slightly slower thanexpected, based on the combined molecular weights of Vpr1 (14.5 kDa) andSN (16 kDa) and Vpx2 (15 kDa) and SN, respectively. Transfection ofpTM-vpr1 and pTM-vpx2 alone yielded appropriately sized wild-type Vprand Vpx proteins. Anti-Vpr, anti-Vpx and anti-SN antibodies were notreactive with lysates of pTM1 transfected cells included as controls.Thus, both SN and SN* fusion proteins can be expressed in mammaliancells.

Example 12 Incorporation of Vpr1- and Vpr2-SN/SN* Fusion Proteins intoVirus-Like Particles

In vaccinia and baculovirus systems, the expression of HIV Gag issufficient for assembly and extracellular release of VLPs. Vpr1 and Vpx2can be efficiently incorporated into Gag particles without theexpression of other viral gene products. To demonstrate that the Vpr1and Vpx2 fusion proteins could be packaged into VLPs, recombinantplasmids were coexpressed with HIV-1 and HIV-2 Gag proteins in the rVT7system. pTM-vpr1, pTM-vpr1SN and pTM-vpr1SN* were transfected into HeLacells alone and in combination with the HIV-1 Gag expression plasmid,pTM-gag1. Twenty-four hours after transfection, cell and VLP extractswere prepared and analyzed by immunoblot analysis (FIG. 3A). Anti-Vprantibody detected Vpr1, Vpr1SN and Vpr1SN* in cell lysates (top panel)and in pelleted VLPs derived by coexpression with pTM-gag1 (middlepanel). In the absence of HIV-1Gag expression, Vpr1 and Vpr1SN were notdetected in pellets of culture supernatants (middle panel). As expectedVLPs also contained p55 Gag (bottom panel). Thus, Vpr1SN/SN* fusionproteins were successfully packaged into VLPs.

To demonstrate that Vpx2SN was similarly capable of packaging into HIV-2VLPs, pTM-vpx2, pTM-vpx2SN and pTM-vpx2SN* were transfected into HeLacells alone and in combination with the HIV-2 Gag expression plasmid,pTM-gag2. Western blots were prepared with lysates of cells and VLPsconcentrated from culture supernatants by ultracentrifugation (FIG. 3B).Anti-Vpx antibody detected Vpx2, Vpx2SN and Vpx2SN* in cell lysates (toppanel) and in VLPs derived by coexpression with pTM-gag2 (middle panel).Anti-Gag antibody detected p55 Gag in VLP pellets (bottom panel).Comparison of the relative protein signal intensities suggested that theVpr1 and Vpx2-SN and SN* fusion proteins were packaged into VLPs inamounts similar to wild-type Vpr1 and Vpx2 proteins. Sucrose gradientanalysis of VLPs containing Vpr1 SN and Vpx2SN demonstratedco-sedimentation of these fusion proteins with VLPs (data not shown).

The Gag C terminal region is required for incorporation of Vpr1 and Vpx2into virions. However, packaging was found to be virus type-specific,that is, when expressed in trans, Vpx2 was only efficiently incorporatedinto HIV-2 virions and HIV-2 VLPs. Similarly, HIV-1 Vpr requiredinteraction with the HIV-1 Gag precursor for incorporation into HIV-1VLPs. To show that the association of Vpr1SN and Vpx2SN with VIPs wasnot mediated by the SN moiety, but was due to the Vpr and Vpx specificpackaging signals, pTM-vpr1SN and pTM-vpx2SN were cotransfectedindividually with either pTM-gag1 or pTM-gag2. For control, pTM-vpr1 andpTM-vpx2 were also transfected alone. Twenty-four hours later, lysatesof cells and pelleted VLPs were examined by immunoblotting (FIG. 4).While Vpr1SN was expressed in all cells (FIG. 4A, top panel), it wasonly associated with VLPs derived from cells transfected with pTM-gag1.Similarly, Vpx2SN was detected in all pTM-vpx2 transfected cells (FIG.4B, top panel), but was only associated with VLPs derived bycotransfection with pTM-gag2 (FIG. 4B, middle panel). HIV-1 and HIV-2Gag monoclonal antibodies confirmed the presence of Gag precursorprotein in each VLP pellet (FIG. 4B, bottom panels). Thus, incorporationof Vpr1SN and Vpx2SN into VLPs requires interaction of the cognate Gagprecursor protein, just like native Vpr1 and Vpx2.

While Vpr1SN and Vpx2SN fusion proteins clearly associated with VLPs(FIG. 3), the question remained whether they would continue to do so inthe presence of the native accessory proteins. The efficiency of Vpr1SNand Vpx2SN packaging was compared by competition analysis (FIG. 5).pTM-vpr1SN and pTM-vpx2SN were cotransfected with pTM-gag1/pTM-vpr1 andpTMgag2/pTM-vpx2, respectively, using ratios that ranged from 1:4 to 4:1(FIG. 5A and FIG. 5B, left panels). For comparison, pTM-vpr1 SN andpTM-vpr1 were transfected individually with pTM-gag1 (FIG. 5A, middleand right panels respectively) and pTM-vpx2SN and pTM-vpx2 weretransfected with pTM-gag2 (FIG. 5B, middle and right panelsrespectively). VLPs were pelleted through sucrose cushions, lysed,separated by PAGE, blotted onto nitrocellulose and probed with anti-SNantibody. The results revealed the presence of both Vpr1 and Vpr1SN inVLPs when cotransfected into the same cells (FIG. 5A, left panel).Similarly, coexpressed Vpx2 and Vpx2SN were also copackaged (FIG. 5B,left panel). Comparison of the relative amounts of VLP-associated Vpr1SNand Vpx2SN when expressed in the presence and absence of the nativeprotein, indicated that there were no significant packaging differences.Thus, Vpr1/Vpx2 fusion proteins can efficiently compete with wild-typeproteins for virion incorporation.

Example 13 Vpr1 SN and Vpx2SN Fusion Proteins Possess Nuclease Activity

To demonstrate that virion associated SN fusion proteins wereenzymatically active, VLPs concentrated by ultracentrifugation fromculture supernatants of HeLa cells transfected with pTM-gag1/pTM-vpr1SNand pTMgag2/pTM-vpx2SN were analyzed for nuclease activity using an invitro DNA digestion assay. Prior to this analysis, immunoblottingconfirmed the association of Vpr1 SN and Vpx2SN with VLPs (data notshown). FIG. 6 shows lambda phage DNA fragments in 0.8% agarose gelsafter incubation with dilutions of VLPs lysates that contained Vpr1- orVpx2-SN fusion proteins. VLPs containing Vpr1 SN* and Vpx2SN* wereincluded as negative controls and dilutions of purified SN served asreference standards (FIG. 6A). Both virion associated Vpr1SN (FIG. 6B)and Vpx2SN (FIG. 6C) fusion proteins exhibited nuclease activity asdemonstrated by degradation of lambda phage DNA. Cell-associated Vpr1SNand Vpx2SN fusion proteins also possessed nuclease activity whenanalyzed in this system (data not shown). To control for SN specificity,this analysis was also conducted in buffers devoid of Ca⁺⁺ and underthese conditions no SN activity was detected (data not shown). Thus, SNremains enzymatically active when expressed as a fusion protein andpackaged into VLPs.

Example 14 Incorporation of Vpx2SN Fusion Protein into HIV-2 Virions

Vpx is incorporated into HIV-2 virions when expressed in trans. To showthat Vpx2 fusion proteins were similarly capable of packaging intowild-type HIV-2 virions, an expression plasmid (pLR2P) was constructedplacing the vpx2SN and vpx2SN* coding regions under control of HIV-2 LTRand RRE elements. The HIV-2 RRE was positioned downstream of the fusiongenes to ensure mRNA stability and efficient translation (FIG. 7A). Toshow that the fusion proteins could package when expressed in trans,HIV-2_(ST), proviral DNA (pSXBI) was transfected alone and incombination with pLR2P-vpx2SN and pLR2P-vpx2SN*. Forty-eight hourslater, extracellular virus as pelleted from culture supernatants byultracentrifugation through cushions of 20% sucrose and examined byimmunoblot analysis (FIG. 7B). Duplicate blots were probed usinganti-Vpx (left), anti-SN (middle) and anti-Gag (right) antibodies.Anti-Vpx antibody detected the 15 kDa Vpx2 protein in all viral pellets.In virions derived by cotransfection of HIV-2_(ST) with pLR2P-vpx2SN andpLR2P-vpx2SN*, additional proteins of approximately 35 and 32 kDa wereclearly visible. The same two proteins were also apparent on a duplicateblot probed with anti-SN antibodies, indicating that they representedVpx2SN fusion proteins (FIG. 7B, middle panel). The predicted molecularweight of full-length Vpx2SN fusion protein is 33 kDa. As native Vpx andSN run slightly slower than predicted, it is likely that the 35 kDaspecies represents the full-length Vpx2SN fusion protein. Anti-SNantibodies detected additional proteins of approximately 21 and 17 kDa(these proteins were more apparent after longer exposure). Since onlythe 35 kDa protein was detected in Gag derived VLPs, which lack Polproteins (FIG. 2), the smaller proteins represented cleavage products ofVpx2SN and Vpx2SN* generated by the viral protease. Anti-Gag antibodiesconfirmed the analysis of approximately equivalent amounts of virionsfrom each transfection.

To show packaging of Vpx2SN into HIV-2 virions, sucrose gradientanalysis was performed. Extracellular virus collected from culturesupernatants of HLtat cells forty-eight hours after cotransfection withpLR2P-vpx2SN and HIV-2_(ST) was pelleted through cushions of 20%sucrose. Pellets were resuspended in PBS and then centrifuged for 18hours over linear gradients of 20-60% sucrose. Fractions were collectedand analyzed by immunoblotting (FIG. 7C). Duplicate blots were probedseparately with anti-SN (top) and anti-Gag (bottom) antibodies. Peakconcentrations of both Vpx2SN and Gag were detected in fractions 8-11,demonstrating direct association and packaging of Vpx2SN into HIV-2virions. These same sucrose factions (8-11) were found to have densitiesbetween 1.16 and 1.17 g/ml, as determined by refractometric analysis(data not shown). Again, both the 35 kDa and 32 kDa forms of Vpx2SN weredetected, providing further evidence for protease cleavage followingpackaging into virus particles.

Since HIV-2_(ST) is defective in Vpr, this may have affected thepackaging of the Vpx2SN fusion protein. A second strain of HIV-2, termedHIV-2_(7312A), was analyzed which was cloned from short-term PBMCculture and contains open reading frames for all genes, including intactvpr and vpx genes (unpublished). A plasmid clone of HIV-2_(7312A)proviral DNA (pJK) was transfected alone and in combination withpLR2P-vpx2SN into HLtat cells. For comparison, HIV-2_(ST) was alsoco-transfected with pLR2P-vpx2SN. Progeny virus was concentrated byultracentrifugation through sucrose cushions and examined by immunoblotanalysis (FIG. 7D). Duplicate blots were probed with anti-Vpx (left) andanti-Gag (right) antibodies. The results revealed comparable levels ofVpx2SN incorporation into vpr competent virus (HIV-2_(7312A)) comparedwith vpr-defective virus (HIV-2_(ST)). Moreover, the 35 kDa and 32 kDaproteins were again detected in HIV-2_(7312A) virions. Thus, efficientincorporation of the Vpx2SN protein into replication-competent wild-typeHIV-2 was demonstrated, even in the presence of native Vpr and Vpxproteins.

Example 15 Incorporation of Vpr1SN into HIV-1 Virions

Using the same LTR/RRE-based expression plasmid, it was also shown thatVpr1SN could package into HIV-1 virions by co-expression with HIV-1provirus (as discussed above, the HIV-2 LTR can be transactivated byHIV-1 Tat and the HIV-2 RRE is sensitive to the HIV-1 Rev protein).Virions released into the culture medium 48 hours after transfection ofHLtat cells with pNL4-3 (HIV-1) and pNL4-3-R⁻ (HIV-1-R⁻) alone and incombination with pLR2P-vpr1SN were concentrated by ultracentrifugationand examined by immunoblot analysis (FIG. 8). As observed incotransfection experiments with HIV-2, anti-SN antibodies identified twomajor Vpr1SN fusion proteins of approximately 34 to 31 kDa. Theseproteins were not detected in virions produced by transfection of pNL4-3and pNL4-e-R⁻ alone. From expression in the rVT7 system, the full-lengthVpr1SN fusion protein was expected to migrate at 34 kDa. Therefore, the31 kDa protein likely represents a cleavage product. Anti-SN antibodiesalso detected a protein migrating at 17 kDa. Anti-Vpr antibody detectedthe 34 and 31 kDa proteins in virions derived from cotransfected cells.It is noteworthy that both the anti-Vpr and anti-SN antibodies detectedthe 31 kDa protein most strongly, and that anti-Vpr antibody did notdetect the 17 kDa protein recognized by anti-SN antibody. These resultsalso show that even in virions in which native Vpr protein was packaged,Vpr1SN was also incorporated in abundance. Gag monoclonal antibodydetected similar amounts of Gag protein in all viral pellets anddemonstrated processing of the p55^(Gag) precursor (FIG. 8C).

To demonstrate more directly that cleavage of the Vpr1- and Vpx2-SNfusion proteins was mediated by the HIV protease, virus was concentratedfrom pNL4-3-R⁻/pLR2P-vpr1SN and pSXB1/pLR2P-vpx2SN transfected cellsthat were culture in the presence of 1 μM of the HIV protease inhibitorL-689,502 (provided by Dr. E. Emini, Merck & Co. Inc.). As expected,immunoblot analysis of virions demonstrated substantially lessprocessing of p55^(Gag) (FIG. 9A). Similarly, virions produced in thepresence of L-689,502 also contained greater amounts of the uncleavedspecies of Vpr1SN and Vpx2SN fusion proteins (FIG. 9B). Taken together,these results demonstrate that Vpr1- and Vpx2-SN fusion proteins aresubject to protease cleavage during or subsequent to virus assembly.

Example 16 Vpr1-CAT and Vpr2-CAT Fusion Protein Incorporation into HIVVirions

To show that Vpx2 and Vpr1 could target additional proteins to the HIVparticle, the entire 740 bp cat gene was substituted for sn in thepLR2P-vpx2SN and pLR2P-vpr1SN vectors, generating pLR2P-vpr1 CAT andpLR2P-vpx2CAT (FIG. 10A). pNL4-3/pLR2P-vpr1 CAT, pn14-3-R⁻/pLR2P-vpr1CAT and pSXB1/pLR2P-vpx2CAT were co-transfected into HLtat cells. Ascontrols, pNL4-3, pNL4-3-R⁻ and pSXB1 were transfected alone. Progenyvirions, concentrated from culture supernatants, were analyzed byimmunoblotting (FIGS. 10B and 10C). Using anti-Vpr antibodies, 40 kDafusion proteins were detected in viral pellets derived byco-transfection of pRL2P-vpr1 CAT with both pNL4-3 and pNL4-3-R⁻ (FIG.10B). This size is consistent with the predicted molecular weight of thefull-length Vpr1 CAT fusion protein. In addition, anti-Vpr antibodiesalso detected a 17 kDa protein which did not correspond to the molecularweight of native Vpr1 protein (14.5 kDa in virions derived from cellstransfected with pNL4-3). The same protein was recognized weakly withanti-CAT antibodies, suggesting a fusion protein cleavage productcontaining most Vpr sequence. Very similar results were obtained withvirions derived from HLtat cells co-transfected with HIV-2_(ST) andpRL2P-vpx2CAT, in which anti-Vpx antibody detected 41 and 15 kDaproteins (FIG. 10C). These results demonstrate that Vpr1 CAT and Vpx2CATfusion proteins are packaged into virions. However, like in the case ofSN fusion proteins, CAT fusion proteins were also cleaved by the HIVprotease (the Vpx2CAT cleavage product is not visible because ofco-migration with the native Vpx protein. CAT cleavage appeared lessextensive, based on the intensity of the full-length CAT fusion proteinon immunoblots.

Lysates of HIV-1 and HIV-2 viral particles were diluted 1:50 in 20 mMTris-base and analyzed for CAT activity by the method of Allon et al.(1979) Nature 282:864-869. FIG. 10D indicates that virions whichcontained Vpr1CAT and Vpx2CAT proteins possessed CAT activity. Theseresults show the packaging of active Vpr1- and Vpx2-CAT fusion proteins.

Example 17 Virion Incorporated SN and CAT Fusion Proteins areEnzymatically Active

The ability of Vpr1 and Vpx 2 to deliver functionally active proteins tothe virus particle was further confirmed by sucrose gradient analysis.Virions derived from HLtat cells co-transfected with HIV-2_(ST) andpLR2P-vpx2 were sedimented in linear gradients of 20-60% sucrose asdescribed above. Fractions were collected and analyzed for viral Gagprotein (FIG. 11) and corresponding CAT activity (FIG. 11B). Peakamounts of Gag protein were detected in fractions 6 and 7 (density 1.16and 1.17, respectively). Similarly, peak amounts of acetylatedchloramphenicol (Ac-cm) were also detected in fractions 6 and 7.

Whether virion associated SN fusion protein retained nuclease activitywas also shown. HIV-1_(SG3) virions containing Vpr1SN were analyzedafter sedimentation in linear gradients of sucrose (FIG. 11). Since thepresent invention demonstrated that protease cleavage of SN fusionproteins (FIGS. 7, 8 and 9) markedly reduced Vpr1SN nuclease activity(data not shown), these experiments were performed by culturingpSG3/pLR2P-vpr1SN co-transfected cells in the presence of L-689,502 asdescribed above. Immunoblot analysis of sedimented virions revealed peakconcentrations of Gag in fractions 6 and 7 and substantially reduced p55processing (FIG. 11C). Peak SN activity was associated with thefractions that contained the highest concentrations of virus (FIG. 11D).These results thus document that virion incorporation per se does notabrogate the enzymatic activity of Vpr/Vpx fusion proteins, althoughcleavage by the viral protease may inactivate the fusion partners.

Example 18 Construction and Design of a Gag-Pro (RT-IN Minus) PackagingPlasmid

Several different strategies have been used to express Gag-Pro. PlacingGag and Pro in the same reading frame leads to overexpression of Pro andmarked cell toxicity. It is known that deletions within the RT and INcoding regions, including smaller deletion mutations, may cause markeddefects in the expression levels of the Gag-Pro and Gag-Pol proteins,respectively (Ansari-Lari et al. (1995) Virology 211:332-335;Ansari-Lari et al. (1996) J. Virol. 70:3870-3875; Bukovsky et al. (1996)J. Virol. 70:6820-6725; Engelman et al. (1995) J. Virol. 69:2729-2736;Schnell et al. (1997) Cell Press 90:849-857). Importantly, the viralparticles produced under these circumstances are defective inproteolytic processing and are not infectious, even if RT and IN areprovided in trans (Wu et al. (1994) J. Virol. 68:6161-6169). The reducedlevels of expression and virion associated Gag-Pol protein is apparentlydue to an effect on the frequency of Gag-Pol frame-shifting. Gag-Polframe-shifting is not markedly affected when the translation of RT andIN is abrogated, which is distinct from deletions of viral DNA fragment.Virions which assembly Gag-Pro, when RT and IN protein synthesis isabrogated by a translational stop codon, mature and are infectious whenRT and IN are provided in trans (Wu et al. (1994) J. Virol.,68:6161-6169). Therefore, a Gag-Pro packaging plasmid of the presentinvention is preferably constructed by abrogating translation ofsequence downstream of Pro (RT-IN). Other mutations in Gag and Pol wouldalso function as part(s) of the trans-lentiviral packaging system ifthey did not cause major defects in particle assembly and infectivity.In addition to introducing a translational stop codon (TAA) at the firstamino acid residue of RT, at least one addition “fatal” mutation ispositioned within RT and IN (FIG. 12B). This mutation further decreasesthe likelihood of reestablishing a complete Gag-Pol coding region bygenetic recombination between packaging (gag-pro) and enzymatic(vpr-RT-IN) plasmids. It is appreciated that the stop codon can beinserted within the gene sequence in a position other than at the firstcodon for the first amino acid residue of a protein and still be aneffective measure to prevent infectivity. A stop codon generallyinserted with the front half of the amino acid encoding nucleic acidresidues is effective, although the stop codon is preferentially at thebeginning of the translational sequence. A fatal mutation as used hereinrefers to a mutation within the gene sequence that render the codedpolypeptide sequence functionally ineffectual in performing thebiological role of the wild protein.

The Gag-Pro expression plasmid (pCR-gag-pro) includes the CMV promoterand the HIV-2 Rev responsive element (RRE) (FIG. 12C). The RRE allowsfor the efficient expression of HIV proteins (including Gag, PR, RT, IN)that contain mRNA inhibitory sequences. RT and IN are provided bytrans-expression with the pLR2P-vpr-RT-IN expression plasmid (FIG. 12C).This vector expresses the Vpr-RT-IN fusion protein which is incorporatedinto HIV-1 virions/vector in trans, and is proteolytically processed bythe viral protease to generate functional forms of RT (p51 and p66) andIN (Wu et al. (1994) J. Virol. 68:6161-6169). This earlier work showsthat functional RT and IN can be provided separately (Vpr-RT and Vpr-IN)(Liu et al. (1997) J. Virol. 71:7704-7710 and Wu et al. (1994) J. Virol.68:6161-6169). Preferably, the Vpr component of the fusion proteincontains a His71Arg substitution which knocks out the Vpr cell cyclearrest function.

Example 19 Production of the Trans-Lentiviral Vector

4 μg each of pCR-gag-pro, pLR2P-vpr-RT-IN (enzymatic plasmid),pHR-CMV-β-gal (marker gene transduction plasmid) and pCMV-VSV-G (envplasmid were transfected into 293T cell line. 293T cells were used sincethey produce high titered stocks of HIV particles/vector and areexquisitely sensitive to transfection, including multiple plasmidtransfections. As a control, in side-by-side experiments, the pΔ8.2packaging plasmid was also transfected with pHR-CMV-β-gal and pCMV-VSV-G(FIG. 12B). The pΔ8.2 plasmid is a lentivirus packaging vector obtainedfrom Dr. D. Trono. The pΔ8.2 produces high titered vector stocks upontransfection with pHR-CMV-β-gal and pCMV-VSV-G (Naldini et al. (1996)Science 272:263-267 and Zhang et al. (1993) Science 259:234-238),(approximately 1-5×10⁵ infectious particles/ml supernatant, with a p24antigen concentration of 150-800 ng/ml). Approximately 72 hours aftertransfection, the culture supernatants were harvested, clarified bylow-speed centrifugation, filtered through a 0.45 micron filter, andanalyzed for p24 antigen concentration by ELISA. To examine the titer ofthe trans-lentiviral vector, supernatant stocks of 25, 5, 1, and 0.2 μlwere used to infect cultures of HeLa cells and IB3 cells. Two dayslater, the cells are stained with X-gal, and positive (blue) cells arecounted using a light microscope. Table 3 shows the titer oftrans-lentiviral vector. These results show that the trans-lentiviralvector can achieve titers as high as 2×10⁵/ml, although its titer isconsistently lower than that of lentiviral vector (2-5 folds less). Fordirect examination of transduction in living cells the transductionplasmid was also constructed to contain the GFP gene/marker (FIGS. 12Band 12C). Stocks of trans-lentiviral and lentiviral vector were producedas described above and used to infect HeLa cells. Two days later thecells were examined by fluorescence microscopy. FIGS. 13 and 14 showpositive gene transduction with the trans-lenti and lentiviral vectorsrespectively.

TABLE 3 Generation of Trans-Lentiviral Vector Titer (inf. units/ml ×10⁻⁵) Packaging Plasmid RT-IN Plasmid HeLA IB3 pCMVΔR9 — 2.5 (+/−5.1)1.2 (+/−2.7) pCMVΔR9-S^(RT-IN) — 0 0 pCMVΔR9-S^(RT-IN) Vpr-RT-IN 1.1(+/−3.1) 0.8 (+/−2.5)

Example 20 Concentration of Trans-Lentiviral Vector byUltracentrifugation

To examine whether the trans-lentiviral vector was stable during theconcentration by ultracentrifugation, the supernatant-trans-lentiviralvector was concentrated by ultracentrifugation (SW28, 23,000 rpm, 90min., 4° C.). As a control supernatant-lentiviral vector wasconcentrated in parallel. The titers for both were determined bothbefore and after concentration. Table 4 shows our results and indicatesthat the trans-lentiviral vector is stable during concentration byultracentrifugation.

TABLE 4 Concentration of Trans-Lentiviral Vector Titer (inf. units/ml ×10⁻⁵) Packaging Plasmid RT-IN Plasmid HeLA IB3 pCMVΔR9 — 54 31pCMVΔR9-S^(RT-IN) Vpr-RT-IN 28 19

Example 21 Trans-Lentiviral Vector for CFTR Gene Transduction

Lentiviral-based vectors are attractive for use in the lung due to theirability to transduce non-divided cells. This unique characteristic mayrepresent an important advantage of lentiviral vectors for gene therapyof CF. A translentiviral vector was used to deliver the CFTR gene intoHeLa cells. The CFTR gene was cloned into the pHR transduction plasmid,using SmaI and XhoI sites (FIG. 15). Trans-lentiviral and lentiviral (ascontrol) vectors were generated by transduction as described above, andused to transduce HeLa cells grown on cover slips. Two days later thecells were examined by immunofluorescence microscopy, using bothpolyclonal (FIG. 16) and monoclonal antibodies (FIG. 17). The resultsshow CFTR expression and localization of CFTR on the cell surface.Furthermore, the transduced HeLa cells examined by SPQ (halide-sensitivefluorophore) showed restored CFTR function (FIG. 18).

The present invention demonstrated the capability of HIV-1 Vpr and HIV-2Vpx to direct the packaging of foreign proteins into HIV virions whenexpressed as heterologous fusion molecules. The trans complementationexperiments with HIV proviral DNA revealed that Vpr1 and Vpx2 fusionproteins were also incorporated into replication-competent viruses.Moreover, packaging of the fusion proteins in the presence of wild-typeVpx and/or Vpr proteins (FIGS. 16 and 17) indicated that the viralsignals mediating their packaging were not obstructed by the foreigncomponents of the fission molecules. Likewise, virion-associated SN andCAT fusion proteins remained enzymatically active.

Based on the immunoblot analysis of VLPs and virions, the presentinvention illustrates that both virion associated CAT and SN/SN* aresusceptible to cleave by the viral protease. There appears to be atleast one cleavage site in CAT and two cleavage sites in the SN/SN*proteins. Based on calculated molecular weights of the major SN/SN*cleavage products, it appears that SN and SN* are cleaved one near theirC termini and once near the fusion protein junctions. Since the fusionprotein junctions of Vpr1SN and Vpx2SN are not identical it is alsopossible that these regions differ with respect to their susceptibilityto the viral protease. Although Vpx2SN/SN* were processed to a lesserextent than Vpr1SN (FIGS. 7 and 8), the major cleavage sites appear tobe conserved. There is no doubt that both the HIV-1 and HIV-2 proteasesrecognize processing sites in the fusion partners and that there issufficient physical contact to enable cleavage. This is evidenced bothby the reduction of cleavage product intensities on immunoblots as wellas by an increased enzymatic activity in the presence of an HIV proteaseinhibitor.

The demonstration that Vpr1 and Vpx2 fusion proteins are capable ofassociating with both VLPs and virions facilitates studies on theseaccessory proteins and on HIV assembly in general. The approach ofgenerating deletion mutants to study protein structure/functionrelationships is often of limited value since this can reduce proteinstability or change the three-dimensional structure of the protein. Inthe case of Vpr, a single amino acid substitution at residue 76 has beenshown to destabilize its expression in infected cells. Studies haveindicated that deletion mutations in vpr and vpx result in prematuredegradation of the proteins following expression. Fusion of Vpr and Vpxmutant proteins with, e.g., SN or CAT as demonstrated by the presentinvention, increase stability.

The successful packaging of Vpr1/Vpx2SN fusion proteins into virionsindicates their use for accessory protein targeted viral inactivation.The present invention demonstrates that Vpr and Vpx may serve asvehicles for specific targeting of virus inhibitory molecules, includingSN. In contrast to HIV Gag, Vpr and Vpx are small proteins that can bemanipulated relatively easily without altering virus replication andthus may represent vehicles with considerable versatility forapplication to such an antiviral strategy.

Example 22 Incorporation of RT in trans into a Lentivirus Independent ofHIV Accessory Proteins

The HIV accessory proteins, Vpr and Vpx, are incorporated into virionsthrough specific interactions with the p6 portion of the Pr55^(Gag)precursor protein (Kappes et al. 1993; Kondo et al. (1995) J. Virol.69:2759-2764; Lu et al. (1995) J. Virol. 69:6873-6879; Paxton et al.(1993) J. Virol. 67:7229-7237; Wu et al. (1994) J. Virol. 68:6161-6169).Similarly, it has been demonstrated that Vpr and Vpx fusion proteins(Vpr- and Vpx-SN and CAT) are incorporated into virions throughinteractions with p6^(Gag), similar to that of the wild-type Vpr and Vpxproteins (Wu et al. (1995) J. Virol. 69:3389-3398). To analyze thecontribution of Vpr for incorporation of the Vpr-RT fusion protein intovirions, an HIV-1 proviral clone mutated in p6^(Gag) and PR (designatedpNL43-Δ p6^(Gag), provided by Dr. Mingjun Huang) was cotransfected withpLR2P-vprRT into 293T cells. This mutant contains a TAA translationalstop colon at the first amino acid residue position of p6^(Gag). Thisabrogated the Gag sequences that are required for Vpr virionincorporation. The pNL43-Δ p6^(Gag) clone also contains a mutation(D25N) in the active site of PR, which enhances the release of thep6^(Gag) mutant virus from the cell surface membrane (Gottlinger et al.1991; Huang et al. 1995). As a control, the HIV-1 PR mutant PM3 (Kohl etal. 1988), derived from the same pNL4-3 parental clone, was alsoincluded for analysis. Progeny virions, purified from pNL43-Δ p6^(Gag)transfected cell cultures, contained detectable amounts of RT protein(labeled as Vpr-p66), albeit in lesser amounts compared with virionsderived from PM3 (FIG. 19). Analysis of cell lysates confirmedexpression, and compared with PM3, the accumulation of Vpr-RT inpLR2P-vprRT/pNL43-Δ p6^(Gag) cotransfected cells. Vpr^(S)-RT wasincluded as an additional control and was shown to incorporate Vprefficiently into PM3 virions but not into those derived by coexpressionwith pNL43-Δ p6^(Gag). Wild-type Vpr protein was also absent from Δp6^(Gag) virions. Approximately equal amounts of Gag protein wasdetected in the different virus pellets, confirming that similar amountsof the different virions were compared in the quantitation. Theseresults show that RT protein can be incorporated into virionsindependently of Vpr-p6 mediated interaction. These data also indicatethat expression of RT (and IN by inference) in trans, independently ofGag-Pol, is sufficient for its incorporation and function.

Example 23 Expression of RT in trans in a Lentivirus Vector Independentof HIV Accessory

It has been demonstrated that functional RT can be incorporated intoHIV-1 virions by its expression in trans, even without fusion to Vpr(Example 19). To determine if RT expressed in trans can package intolentiviral vector and support the transduction of a marker gene RT wasligated into the pLR2P expression plasmid under control of the HIV LTRand RRE, generating the pLR2P-RT expression plasmid. The pLR2P-RT,pHR-CMV-VSV-G, pHR-CMV-β-gal, and pΔ8.2-RT^(D185N) was transfectedtogether into 293T cells. The pΔ8.2-RT^(D185N) plasmid contains a pointmutation in RT at amino acid residue position 185 (D185N), whichabolishes polymerase activity and destroys its ability to support genetransduction. As a control Vpr-RT (pLR2P-vpr-RT) was substituted forpLR2P-RT in a parallel experiment. As another control neither RT orVpr-RT were provided. Virions generated by transfection were used toinfect HeLa cells. Two days later, transduction positive cells werecounted. FIG. 20 shows that both Vpr-RT and RT support vectortransduction when provided in trans. The vector titer was reduced byabout 10-fold when RT was provided without fusion with Vpr. Theseresults demonstrate that enzymatic function (RT and IN) can be providedin trans, independently of Gag-Pol.

The present invention demonstrated that Vpr and Vpx can serve asvehicles to deliver functionally active enzymes to the HIV virion,including those that may exert an antiviral activity such as SN. Thepresent invention has demonstrated that the concept of accessory proteintargeted virus inactivation is feasible.

Any patents or publications mentioned in this specification areindicative of the levels of those skilled in the art to which theinvention pertains. These patents and publications are hereinincorporated by reference to the same extent as if each individualpublication was specifically and individually indicated to beincorporated by reference.

One skilled in the art will readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. The presentexamples along with the methods, procedures, treatments, molecules, andspecific compounds described herein are presently representative ofpreferred embodiments, are exemplary, and are not intended aslimitations on the scope of the invention. Changes therein and otheruses will occur to those skilled in the art which are encompassed withinthe spirit of the invention as defined by the scope of the claims

1. A nucleic acid molecule comprising a first nucleotide sequenceexpressing a Vpr polypeptide fused in frame to a second nucleotidesequence expressing a full length Integrase polypeptide, wherein saidfirst nucleotide sequence expresses a full length Vpr polypeptidecapable of association with an HIV viron, an SIV viron, or a virus likeparticle.
 2. A nucleic acid molecule comprising a first nucleotidesequence expressing a Vpr polypeptide fused in frame to a secondnucleotide sequence expressing a full length Reverse Transcriptasepolypeptide, wherein said first nucleotide sequence expresses a fulllength Vpr polypeptide capable of association with an HIV viron, an SIVviron, or a virus like particle.
 3. The nucleic acid molecule of claim1, wherein said first nucleotide sequence is from a HumanImmunodeficiency Virus or a Simian Immunodeficiency Virus.
 4. Anexpression plasmid comprising a nucleic acid molecule of claim
 1. 5. Anexpression plasmid comprising a nucleic acid molecule of claim
 2. 6. Theexpression plasmid of claim 4, wherein said nucleic acid molecule isoperably linked to a regulatory element necessary for expression of saidnucleic acid molecule in a cell.
 7. The expression plasmid of claim 6,wherein said regulatory element is selected from the group consisting ofan HIV-2 RRE and an HIV-2 LTR.
 8. The expression plasmid of claim 6,wherein said regulatory element comprises a promoter capable of drivingexpression in a cell.
 9. A cell having the nucleic acid molecule ofclaim
 1. 10. A cell having the nucleic acid molecule of claim
 2. 11. Thecell of claim 9, wherein said cell is selected from the group consistingof a bacterial cell, a mammalian cell, and an insect cell.